Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus‐infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) ‐A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1‐specific cytotoxic T‐lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT‐specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA‐A2‐restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT‐specific CTL clones did not lyse EBV‐carrying lymphoblastoid cell lines and Burkitt’s lymphoma cell lines nor EBNA1‐transfected Burkitt’s lymphoma cells but specifically released interferon‐γ upon stimulation with HLA‐matched EBNA1‐expressing cells and this response was enhanced by deletion of the Gly‐Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.     

Allelic Diversity and Naturally Acquired Allele-Specific Antibody Responses to Plasmodium falciparum Apical Membrane Antigen 1 in Kenya

Although Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate, extensive allelic diversity may compromise its vaccine potential. We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in this Kenyan population. To assess the impact of allelic diversity on naturally acquired immunity, we first sequenced the ectodomain-encoding region of P. falciparum ama1 from subjects with asymptomatic, mild, and severe malaria and measured allele frequency distributions. We then measured antibodies to three allelic AMA1 proteins (AMA1_3D7, AMA1_FVO, and AMA1_HB3) and used competition enzyme-linked immunosorbent assays (ELISAs) to analyze allele-specific antibodies. Seventy-eight unique haplotypes were identified from 129 alleles sampled. No clustering of allelic haplotypes with disease severity or year of sampling was observed. Differences in nucleotide frequencies in clinical (severe plus mild malaria) versus asymptomatic infections were observed at 16 polymorphic positions. Allele frequency distributions were indicative of balancing selection, with the strongest signature being identified in domain III (Tajima''s D = 2.51; P < 0.05). Antibody reactivities to each of the three allelic AMA1 proteins were highly correlated (P < 0.001 for all pairwise comparisons). Although antibodies to conserved epitopes were abundant, 48% of selected children with anti-AMA1 IgG (n = 106) had detectable reactivity to allele-specific epitopes as determined by a competition ELISA. Antibodies to both conserved and allele-specific epitopes in AMA1 may contribute to clinical protection.Many candidate antigens for subunit malaria vaccines are polymorphic in natural populations, posing challenges for vaccine development. It is important to know how many alleles of a particular candidate will need to be included in a vaccine to induce antibodies with specificity broad enough to recognize the existing antigenic diversity. Populations of Plasmodium falciparum in areas where the disease is highly endemic have high recombination rates (13, 37, 41) and can generate additional haplotypic diversity with every meiotic recombination (54). This is exemplified by apical membrane antigen 1 (AMA1), for which numerous distinct haplotypes are observed, particularly in areas with relatively high malaria transmission intensities (15, 20, 44, 45, 51). These haplotypes are comprised of single-nucleotide polymorphisms, which are distributed throughout the single-locus ama1 gene, but are especially numerous in the portion encoding its surface-accessible ectodomain. Independent studies provide strong evidence that balancing selection is acting to maintain these polymorphisms in the population (15, 20, 44, 45), reflecting the importance of AMA1 as a target of protective immunity. These polymorphisms may need to be incorporated into a vaccine based on AMA1. In animal models, immunization confers better protection against challenge with parasites bearing homologous rather than heterologous alleles of AMA1 (16, 29). Likewise, invasion inhibition is more efficient against parasites bearing homologous alleles (21, 27). Recent studies suggested that the allelic diversity in ama1 could be covered by vaccination with a combination of allelic types (27, 30). However, only a few allelic variants can realistically be included in a vaccine formulation, and it remains to be determined how effective this would be in populations where malaria is endemic, where individuals are repeatedly challenged with parasites bearing diverse ama1 alleles. For example, over 200 unique haplotypes of AMA1 were recently reported for a single geographical location in Mali (51).We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in a population in coastal Kenya (42). Here we explore the impact of the allelic diversity of ama1 on naturally acquired antibodies in this population. We compare the allelic diversities observed among parasite isolates obtained from children with asymptomatic infections and mild and severe clinical malaria. We test for signatures of balancing selection acting on the ama1 gene in this population, as reported previously for other populations, and describe antibody responses to proteins representing three allelic versions of AMA1 before, during, and after clinical infections.     

The Regenerative Potential of the Kidney: What Can We Learn from Developmental Biology?

Cell turnover in the healthy adult kidney is very slow but the kidney has a strong capacity for regeneration after acute injury. Although many molecular aspects of this process have been clarified, the source of the newly-formed renal epithelial cells is still being debated. Several studies have shown, moreover, that the repair of injured renal epithelium starts from mature tubular cells, which enter into an activated proliferative state characterized by the reappearance of mesenchymal markers detectable during nephrogenesis, thus pointing to a marked plasticity of renal epithelial cells. The regenerative potential of mature epithelial cells might stem from their almost unique morphogenetic process. Unlike other tubular organs, all epithelial and mesenchymal cells in the kidney derive from the same germ layer, the mesoderm. In a fascinating view of vertebrate embryogenesis, the mesoderm might be seen as a cell layer capable of oscillating between epithelial and mesenchymal states, thus acquiring a remarkable plasticity that lends it an extended potential for innovation and a better control of three-dimensional body organization. The renal papilla contains a population of cells with the characteristic of adult stem cells. Mesenchymal stromal stem cells (MSC) have been found to reside in the connective tissue of most organs, including the kidney. Recent studies indicate that the MSC compartment extends throughout the body postnatally as a result of its perivascular location. Developmental biology suggests that this might be particularly true of the kidney and that the papilla might represent the perivascular renal stem cell niche. The perivascular niche hypothesis fits well with the evolving concept of the stem cell niche as an entity of action. It is its dynamic capability that makes the niche concept so important and essential to the feasibility of regenerative medicine.     

Stromal and epithelial caveolin-1 both confer a protective effect against mammary hyperplasia and tumorigenesis: Caveolin-1 antagonizes cyclin D1 function in mammary epithelial cells

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1-/- mice and performed a series of mammary transplant studies, using both wild-type and Cav-1-/- mammary fat pads. Cav-1-/- mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1-/- mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis.     

Caveolin-1 deficiency (-/-) conveys premalignant alterations in mammary epithelia, with abnormal lumen formation, growth factor independence, and cell invasiveness

During breast cancer development, the luminal space of the mammary acinar unit fills with proliferating epithelial cells that exhibit growth factor-independence, cell attachment defects, and a more invasive fibroblastic phenotype. Here, we used primary cultures of mammary epithelial cells derived from genetically engineered mice to identify caveolin-1 (Cav-1) as a critical factor for maintaining the normal architecture of the mammary acinar unit. Isolated cultures of normal mammary epithelial cells retained the capacity to generate mammary acini within extracellular matrix. However, those from Cav-1 (-/-) mice exhibited defects in three-dimensional acinar architecture, including disrupted lumen formation and epidermal growth factor-independent growth due to hyperactivation of the p42/44 mitogen-activated protein kinase cascade. In addition, Cav-1-null mammary epithelial cells deprived of exogenous extracellular matrix underwent a spontaneous epithelial-mesenchymal transition, with reorganization of the actin cytoskeleton, and E-cadherin redistribution. Mechanistically, these phenotypic changes appear to be caused by increases in matrix metalloproteinase-2/9 secretion and transforming growth factor-beta/Smad-2 hyperactivation. Finally, loss of Cav-1 potentiated the ability of growth factors (hepatocyte growth factor and basic fibroblast growth factor) to induce mammary acini branching, indicative of a more invasive fibroblastic phenotype. Thus, a Cav-1 deficiency profoundly affects mammary epithelia by modulating the activation state of important signaling cascades. Primary cultures of Cav-1-deficient mammary epithelia will provide a valuable new model to study the spatial/temporal progression of mammary cell transformation.     

Quantitative distribution of a panel of circulating mRNA in preeclampsia versus controls

OBJECTIVE: The aim of this study was to evaluate whether the quantitative distribution of a panel of circulating mRNAs from maternal whole blood of normal pregnancies is statistically different from those complicated with preeclampsia (PE) with or without intrauterine growth restriction (IUGR). METHODS: Maternal whole blood of six subjects with mild or severe PE with or without IUGR and 30 matched controls (1:5 match for gestational age) were retrospectively examined for circulating mRNA markers. Seven specific mRNA markers were identified and chosen based on previous microarray mRNA expressions performed on placental tissue from normal and PE patients. They were human placental lactogen (hPL), inhibin A, KISS-1, pregnancy-associated plasma protein-A (PAPP-A), plasminogen activator inhibitor type 1 (PAI-1), selectin-P and vascular endothelial growth factor receptor (VEGFR), which were therefore quantified for statistical purposes. RESULTS: Median gestational age was 229 (178-283) and 232 (194-262) days for controls and cases respectively. All mRNA markers but PAPP-A, showed statistically different median values. They were hPL, inhibin A, KISS-1, PAI-1, Selectin-P, and VEGFR. Inhibin A, Selectin-P and VEGFR showed higher values than expected for controls. Instead, hPL, KISS-1 and PAI-1 values of PE patients were lower than those of controls. Selectin-P was the marker with the most aberrant difference, followed by VEGFR and KISS-1. CONCLUSION: This preliminary analysis revealed that the median values of a panel of mRNAs from the maternal blood of PE patients were different from those of the same gestational age control group at the third trimester. If prospective studies at the second trimester could detect a related marker sufficiently able to discriminate between affected and unaffected patients and thus detect the disease before its clinical onset, then a screening project using a panel of mRNAs would be feasible.